Proteomics and peptidomics in the search for (early) biomarkers of asthma

Occupational asthma (OA) is the most common work-related lung disease in the industrialized world and is characterized by reversible airway obstruction, non-specific airway hyperreactivity and airway inflammation. In 90% of the cases, a complaints-free latency period precedes the development of immunologically mediated OA resulting in late diagnosis of these workers. Proteomics and peptidomics techniques could help to find (new) proteins or peptides related to the pathophysiology and onset of OA, leading to novel biomarkers to identify sensitized workers. So far, theuse of these techniques in pulmonary research is limited. The research unit of Lung Toxicology has described a well-characterized and validated mouse model of chemical-induced asthma. Two dermal sensitizations and a single airway challenge with a chemical sensitizer, such as toluene diisocyanate resulted in several phenotypical characteristics of chemical-induced asthma, including lymphocyte proliferation and activation, non-specific airway hyperreactivity and neutrophilic lung inflammation. In this doctoral thesis we focused on:a) applying a proteomics approach to find biomarkers of sensitization caused by chemicals and biomarkers of chemical-induced asthma (Chapter 2 and 3)b) investigating the role of B lymphocytes during sensitization (Chapter 4)c) optimizing a label-free peptidomics approach to study peptide biomarkers (Chapter 5) We used two dimensional difference gel electrophoresis (2D-DIGE) to search for possible biomarkers of sensitization in auricular lymph nodes and serum of mice that were sensitized once andtwice. In the auricular lymph nodes, a large portion of the differentially expressed proteins are structural proteins (e.g. actin, vimentin), while another substantial group are immune related proteins (e.g. lymphocyte specific protein-1, coronin 1a). Most of the identified proteins in serum, after one or two sensitizations are involved in binding/transport (e.g. hemopexin, apolipoprotein E). Three potential biomarkers, lymphocyte specific protein-1, coronin 1a and hemopexin, were verified in an independent experiment by Western blot analysis or ELISA. In a next step, we aim to validate this set of proteins in TDI-exposed workerswith the potential of advancing early diagnosis of OA. To investigate possible biomarkers of disease (chemical-induced asthma), we compared the proteome changes in auricular lymph nodes, bronchoalveolar lavage (BAL) and serum of mice after the establishment of asthma. A majority of differentially expressed proteins (e.g. lymphocyte specific protein-1, vitamin D binding protein, apolipoprotein E, hemopexin and haptoglobin) could be directly linked to inflammation (both locally in the lung as systemically in serum), a hallmark of asthma. For the first time,a systematic proteomics approach was implemented using different samples obtained from the same mice in the setting of chemical-induced asthma.The role of B lymphocytes in the development of chemical-induced asthma is still unclear. Recently, it was shown that B lymphocytes from sensitized mice could carry a sensitized signature compared to B lymphocytes of naïve mice. We isolated B lymphocytes from sensitized and non-sensitized mice and studied this sensitized signature by using 2D-DIGE. The altered expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 in sensitized B lymphocytes could be linked directly to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses. The low molecular weight proteome ( In conclusion, the introduction of proteomics and (in a lesser extent) peptidomics in a mouse model of chemical-induced asthma has led to the discovery of several potential biomarkers of sensitization and disease, encouraging further validation of these results in humans.status: publishe